1. Field of the Invention
The present invention relates to a novel methods and reagents useful for the measurement of calcium ions, in Particular, calcium ions in blood and other physiological fluids. 2 Description of the Prior Art
Calcium is an important electrolyte whose monitoring aids the physician in evaluation of patient's health. Changes in calcium level which usually is relatively stable in most body fluids (in blood from 8.2 to 10.3 mg/dl) may indicate presence of various pathological conditions. Calcium levels significantly lower than normal may indicate hypoparathyroidism, vitamin D deficiency or nephritis. Values greater than normal may indicate hyperparathyroidism, vitamin D intoxication or myeloma. Thus, to detect any disease early, which would be manifested by an abnormal calcium level, the method for calcium measurement has to be very accurate and precise.
A number of chemical and physical procedures are known for the determination of calcium. Direct colorimetric procedures are preferred over tedious precipitation, gravimetric or titrimetric procedures. Generally, such colorimetric procedures involve the complexation of a dye with calcium ions to provide a measurable shift in dye absorption.
A potential problem in the determination of calcium ions in most fluids is the presence of potentially interfering ions (for example, magnesium or iron) or large molecules (for example, proteins or bilirubin). Magnesium ions present a particularly difficult problem because they tend to complex to the same compounds that complex with calcium ions. Accordingly, the prior art has developed procedures whereby magnesium ions can be removed physically from the test sample prior to calcium determination or masked with a magnesium-specific complexing reagent.
Highly selective compounds for calcium ions are described by Tsien in Biochem., 19, pp. 2396-2404 (1980). The parent compound described therein is 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, commonly known as BAPTA. These compounds absorb in the ultraviolet region of the electromagnetic spectrum. This is a significant disadvantage as other species, such as bilirubin, hemoglobin and other porphorin species and metabolic by-products of porphyrins, found in analyte solutions such as blood plasma, spinal fluid, urine and other body fluids, also absorb in the UV and short visible wavelength portions of the electromagnetic spectrum, and produce background interference with standard colorimetric equipment and procedures. Therefore, it was thought desirable to have highly selective calcium complexing compounds which would be detectable at longer wavelengths, and which would shift to other wavelengths when complexed with calcium to allow quantitative analysis for calcium without interference from UV and short wavelength visible light-absorbing species. U.S. Pat. No. 4,795,712 to Toner, et al. describes such calcium complexing dyes. Chromogenic derivatives of 1,2-bis(o-aminoaryloxy)ethane-N,N,N',N'-tetraacetic acid are useful for the determination of calcium ions in both solution and dry assays are disclosed. These compounds comprise a dye moiety which is directly conjugated to the acetic acid substituted-nitrogen atom, and which enables the compounds to exhibit maximum absorbance at a wavelength generally greater than 400 nm before complexation. When the compounds are complexed with calcium ions, the absorbance shifts to a shorter wavelength.
Fluorescent intracellular calcium indicators based on tetracarboxylic compounds are reported in U.S. Pat. No. 4,849,362. The compounds have longer absorption wavelength characteristics which make them useful for multiparameter flow cytometric analysis of intracellular calcium ion concentrations in mixed cell populations.
The orthocresolphthalein complexone method (CPC method), based on a tetracarboxylic acid chelate, first described by A. G. Flaschka, et al., Helv. Chim. Acta 1954, 37, 113, is considered to be the most acceptable calcium method by clinical laboratories. Despite its widespread use, the method suffers from relatively short reagent stability, sensitivity to carbon dioxide and stoichiometry dependent low end non-linearity. Moreover, this method reduces sample throughput (rate of analysis or tests per hour) on automated clinical analyzers. The chromogenic ionophores of the present invention permit the development of a single reagent method with extended stability of the reagent and full range linearity.
A major factor which has to be taken into consideration during the design of possible synthetic candidates for novel calcium chromoionophores is a bathochromic shift of the wavelength maximum upon complexation with calcium. A great number of compounds reported in the literature are derived from 1,2-bis(o-aminophenoxy)ethane-N,N',N'-tetraacetic acid, commonly known a BAPTA. The chromogenic analogs of these structures contain chromophores positioned para to the sensing nitrogen atom, and exhibit a hypsochromic shift of the wavelength maximum upon calcium binding. This in itself is an undesirable characteristic due to the potential spectral interferences present at the shorter wavelengths. In addition, the starting absorbance of the reagent is usually very high.
The CPC method employs a tetracarboxylic acid compound which incorporates two phenol subunits and a chromophore attached oara to the phenolic hydroxyls. Such configuration of the chelating sites and the chromophore system guarantees the bathochromic shift upon complexation with calcium since the cation stabilizes the excited state of the ligand. Thus, in developing new compounds, it would be advantageous to preserve a similar chromophore arrangement, but incorporate into the structure the relatively rigid tetra and triaryl backbones. The tetra and triaryl subunits have successfully been used before in the construction of sodium and potassium selective binders, and promise high degree of preorganization of the chelating sites with respect to the phenolic sensing atoms, forming a polar "nest" complementary with the calcium ion. Novel structures employing carboxylic acid moieties as the chelating groups were chosen.
The compounds of the present invention can generally be described as chromogenic octadentate tetracarboxylic-diphenol and phenol compounds that produce color change in the visible range upon interaction with calcium ions. The chromoionophore is structurally different from other known calcium complexing compounds. It incorporates four (4) acetic acid chelating groups, and one (1) or two (2) nitrophenylazophenyl chromophores attached to a rigid tetraaryl or triaryl framework. The chromogenic compound reacts with calcium ions producing a bathochromic shift and an increase in absorbance at 550 nm in proportion to calcium concentrations.
Accordingly, it has been found that the chromogenic compounds of the present invention demonstrate sensitivity to calcium ions. The chromogenic compounds can be incorporated into a single liquid reagent adapted for use on automated clinical analyzers to determine the calcium concentration in physiological fluid samples such as blood.